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Residues within Transmembrane Helices 2 and 5 of the Human Gonadotropin-Releasing Hormone Receptor Contribute to Agonist and Antagonist Binding

Department of Cancer Research (S.H.H., T.B.) ASTA Medica AG D-60314 Frankfurt/Main, Germany
Department of Molecular Membrane Biology (S.H.H., H.R.) Max-Planck-Institute of Biophysics D-60528 Frankfurt/Main, Germany
Institute of Molecular Pharmacology (T.t.L., R.K.) D-10315 Berlin, Germany

To understand the ligand binding properties of the human GnRH receptor (hGnRH-R), 24 site-specific mutants within transmembrane helices (TMH) 1, 2, and 5 and the extracellular loop 2 (E2) were generated. These mutants were analyzed by using a functional reporter gene assay, monitoring receptor signaling via adenylate cyclase to a cAMP-responsive element fused to Photinus pyralis luciferase. The functional behavior of 14 receptor mutants, capable of G-protein coupling and signaling, was studied in detail with different well described agonistic and antagonistic peptide ligands. Furthermore, the binding constants were determined in displacement binding experiments with the antagonist [¹²⁵I]Cetrorelix.

The substitution of residues K³⁶, Q²⁰⁴, W²⁰⁵, H²⁰⁷, Q²⁰⁸, F²¹⁰, F²¹³, F²¹⁶, and S²¹⁷ for alanine had no or only a marginal effect on ligand binding and signaling. In contrast, substitution of N⁸⁷, E⁹⁰, D⁹⁸, R¹⁷⁹, W²⁰⁶, Y²¹¹, F²¹⁴, and T²¹⁵ for alanine resulted in receptor proteins neither capable of ligand binding nor signal transduction. Within those mutants affecting ligand binding and signaling to various degrees, W¹⁰¹A, N¹⁰²A, and N²¹²Q differentiate between agonists and antagonists. Thus, in addition to N¹⁰² already described, the residues W¹⁰¹ in TMH2 and N²¹² in TMH5 are important for the architecture of the ligand-binding pocket. Based on the experimental data, three-dimensional models for binding of the superagonist D-Trp⁶-GnRH (Triptorelin) and the antagonist Cetrorelix to the hGnRH-R are proposed. Both decapeptidic ligands are bound to the receptor in a bent conformation with distinct interactions within the binding pocket formed by all TMHs, E2, and E3. The antagonist Cetrorelix with bulky hydrophobic N-terminal amino acids interacts with quite different receptor residues, a hint at the failure to induce an active, G protein-coupling receptor conformation.

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